KR20240107344A - Synthetic Methods for Preparation of Modified GCC Receptor Agonists - Google Patents

Abstract

The present invention relates to a method for preparing the synthetic peptide of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof.

Description

Synthetic Methods for Preparation of Modified GCC Receptor Agonists

Cross-reference to related applications

This application claims priority to and the benefit of U.S. Provisional Application No. 63/282,851, filed on November 24, 2021, the contents of which are incorporated herein by reference in their entirety.

Technology field

The present invention relates to a method for preparing the synthetic peptide of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof.

sequence list

This application incorporates by reference the entire Sequence Listing XML in the file name "223355-519433.xml" (8.11 kilobytes) created at 3:45 PM on November 18, 2022 and filed electronically with this application.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic condition that typically involves bladder pain with urinary urgency, increased frequency of urination, and/or nocturia. IC/BPS is often misdiagnosed as a urinary tract infection, for which antibiotics are generally ineffective. It is estimated that 3-7% of women and 3-4% of men meet the definition of IC/BPS. There may be multiple contributing factors to the etiology of IC/BPS, and it is not known whether IC/BPS is a primary disorder or a secondary consequence of another disorder [Hanno et al. 2015, 193; 1545-1553]. There is no diagnostic test for IC/BPS, and the diagnosis is usually based on urinary symptoms of urinary urgency and frequent urination accompanied by pain related to the bladder. Diagnosis is usually withheld until other conditions that may cause these symptoms have been ruled out.

There are few approved therapies available for IC/BPS. Patients often begin treatment with non-pharmacological treatments (general relaxation, stress management, behavior modification, and physical therapy techniques). Because of the minimally effective therapies available for IC/BPS, many patients utilize off-label therapies, including intravesical instillations (i.e., a mixture of medications delivered directly to the bladder via a catheter) to relieve their symptoms. More effective and well-tolerated treatments for IC/BPS are needed.

A 13-amino acid, guanylate cyclase C (GC-C) agonist synthetic peptide is being developed for the treatment of bladder pain associated with IC/BPS, and potentially other visceral pain conditions in the abdominal region. For further development of these peptides, a need exists for efficient synthesis and purification methods.

The present invention relates to a method for producing synthetic peptides or pharmaceutically acceptable salts thereof. The method includes (i) chemically synthesizing a linear peptide having a protected amine group at the N-terminus and a C-terminus bound to a solid phase support using a plurality of amino acids and at least one polyamino acid synthase, wherein the linear The peptide has a protecting group on one or more amino acids and/or at least one polyamino acid unit; wherein the synthetic unit has at least one acetylated amine group and at least one carboxylic acid protecting group; (ii) removing the protecting group from the carboxylic acid protecting group of the synthetic unit and the amine group of the N-terminus of the linear peptide to form a partially unprotected solid phase support-bound peptide having an unprotected amine group and an unprotected carboxylic acid group. steps; (iii) coupling the unprotected amine group and the unprotected carboxylic acid group to form a cyclized solid phase support-linked peptide; (iv) cleaving the cyclized solid-phase support-bound peptide from the solid-phase support to generate a cyclization-protected peptide; (v) globally deprotecting the cyclization protected peptide to obtain a globally deprotected peptide; (vi) folding the generally deprotected peptide to form one or more additional cross-links to obtain a synthetic peptide; and (vii) purifying the synthetic peptide. The synthetic peptide contains the following amino acid sequence: Ac-Cys ₁ Cth ₂ Glu ₃ Leu ₄ Cys ₅ Cys ₆ Asn ₇ Val ₈ Ala ₉ Cys ₁₀ Tyr ₁₁ Gly ₁₂ Cys ₁₃ (SEQ ID NO: 1). The synthetic peptide contains covalent bonds between the following amino acid residues: Cys ₁ and Cys ₆ , Cth ₂ and Cys ₁₀ , and Cys ₅ and Cys ₁₃ .

Figure 1 shows an exemplary flow diagram for the preparation of a linear synthetic peptide of step (i) of the method described herein.

Methods for preparing synthetic peptides or pharmaceutically acceptable salts thereof are described herein. The method described herein includes the following steps:

(i) chemically synthesizing a linear peptide having a protected amine group at the N-terminus and a C-terminus bound to a solid phase support using a plurality of amino acids and at least one polyamino acid synthesis unit, wherein the linear peptide is Having a protecting group on one or more amino acids and/or at least one polyamino acid synthesis unit;

wherein the synthetic unit has at least one acetylated amine group and at least one carboxylic acid protecting group;

(ii) removing the protecting group from the carboxylic acid protecting group of the synthetic unit and the amine group of the N-terminus of the linear peptide, thereby producing a partially unprotected solid phase support-bound peptide having an unprotected amine group and an unprotected carboxylic acid group; forming step;

(iii) coupling the unprotected amine group and the unprotected carboxylic acid group to form a cyclized solid phase support-linked peptide;

(iv) cleaving the cyclized solid-phase support-bound peptide from the solid-phase support to generate a cyclization-protected peptide;

(v) globally deprotecting the cyclization protected peptide to obtain a globally deprotected peptide;

(vi) folding the generally deprotected peptide to form one or more additional cross-links to obtain a synthetic peptide; and

(vii) purifying the synthetic peptide;

wherein the synthetic peptide comprises the following amino acid sequence:

Ac-Cys ₁ Cth ₂ Glu ₃ Leu ₄ Cys ₅ Cys ₆ Asn ₇ Val ₈ Ala ₉ Cys ₁₀ Tyr ₁₁ Gly ₁₂ Cys ₁₃ (SEQ ID NO: 1);

The synthetic peptide herein contains covalent bonds between the following amino acid residues: a) Cys ₁ and Cys ₆ , b) Cth ₂ and Cys ₁₀ , and c) Cys ₅ and Cys ₁₃ .

Justice

As used herein, “Cth” refers to cystathionine, which has two α-amino carboxyl groups, designated “1” and “2” in Scheme 1, and is capable of forming a peptide bond.

However, to facilitate the use of the 3-letter amino acid code to describe peptide sequences, cyclic peptide sequences have each α-amino carboxyl group ("1" and "2") at a non-contiguous position within the peptide sequence. , the peptide bond formed by the α-amino carboxyl group at position 1 is designated “Cth”, while the peptide bond formed by the α-amino carboxyl group at position 2 is designated as “Cth”. The peptide bond formed by the carboxyl group is designated “Cys”. See the section entitled “Synthetic Peptides” for further details.

As used herein, “Hcy” or “Hcys” refers to homocysteine as shown in Scheme 1. As can be seen from Scheme 1, cystathionine can be viewed as a combination of homocysteine and cysteine whose side chains share a sulfur atom. Therefore, an alternative way to designate a cyclic peptide sequence resulting from forming a peptide bond with each α-amino carboxyl group of cystathionine at a non-consecutive position within the peptide sequence is the α-amino carboxyl group at position 1 “Hcy”. It designates a peptide linkage formed by a boxyl group and a peptide linkage formed by an α-amino carboxyl group at position 2 “Cys”.

Unless otherwise indicated, “pharmaceutically acceptable” as used herein means biologically or pharmacologically compatible for use in animals or humans, preferably federally approved for use in animals, more particularly humans. or means approved by a state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia.

As used herein, and unless otherwise indicated, the terms “about” and “approximately” mean within a tolerance for a particular value as determined by one of ordinary skill in the art, which in part means that the value is How it is measured or determined will depend on the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation per implementation in the art. Alternatively, “about” in relation to a composition can mean plus or minus a range of up to 20%, preferably up to 10%. Alternatively, particularly in relation to biological systems or methods, the term can mean within one order of magnitude, preferably within five times, more preferably within two orders of magnitude. Unless otherwise stated, specific values are set forth in this application and claims, and the term “about” means within a tolerance for the specific value.

synthetic peptides

In some embodiments, synthetic peptides made by the methods of the disclosure have the following compounds: Ac-Cys ₁ Cth ₂ Glu ₃ Leu ₄ Cys ₅ Cys ₆ Asn ₇ Val ₈ Ala ₉ Cys ₁₀ Tyr ₁₁ Gly ₁₂ Cys ₁₃ (SEQ ID NO: 1), where “Ac” indicates that the N-terminal amine group is acetylated.

The synthetic peptide of SEQ ID NO: 1 consists of four cysteine residues forming two disulfide bonds, and a cystathione (Cth) unit (side chain sulfur atom) providing an internal sulfide (or thioether) bond with defined connectivity. It contains a combination of homocysteine and cysteine (Cys ₁ -Cys ₆ , Cys ₅ -Cys ₁₃ , Cth ₂ -Cys ₁₀ ).

For the purposes of this description, the two portions of the linear sequence are designated Cth ₂ and Cys ₁₀ , where a thioether bond connects the carbons of the sulfur homocysteine (Hcy) side chain and the des-SH cysteine side chain: the double amino acids are Although it corresponds to a cystathionine (Cth) residue, the proposed designation facilitates description when using the three-letter code designation of the residue, wherein the peptide formed by the α-amino carboxyl group at position 1 of cystathionine The linkage is designated “Cth” and the peptide linkage formed by the α-amino carboxyl group at position 2 is designated “Cys”.

Alternatively, for the purposes of this description, the two parts of the building block may be designated as [Hcy] and [Cys], respectively, where the sulfur of the side chain of homocysteine (Hcy) is shared with the side chain of cysteine (Cys) to form a thio Forms an ether bridge: This double amino acid corresponds to a cystathionine (Cth) residue, but the proposed designation facilitates description when using the 3-letter code designation of the residue. Using this alternative nomenclature, SEQ ID NO: 1 is shown as follows: Ac-Cys ₁ Hcy ₂ Glu ₃ Leu ₄ Cys ₅ Cys ₆ Asn ₇ Val ₈ Ala ₉ Cys ₁₀ Tyr ₁₁ Gly ₁₂ Cys ₁₃ (SEQ ID NO: 1).

In some embodiments, the designation of Cth ₂ -Cys ₁₀ , or any variation thereof, is to describe a linkage between the side chains of two non-consecutive amino acids in SEQ ID NO: 1 forming a thioether bridge as shown below. It is intended:

.

.

(SEQ ID NO: 1) In some embodiments, the designation of Cth ₂ -Cys ₁₀ or any variant thereof describes a cystathionine that forms a peptide bond and forms a thioether bridge at positions 2 and 10 of the synthetic peptide. .

In some embodiments, the synthetic peptide of SEQ ID NO: 1 can be represented by the formula:

.

.

(SEQ ID NO: 1)

Method for preparing synthetic peptides

The method described herein begins by (i) chemically synthesizing a linear peptide having a protected amine group at the N-terminus and a C-terminus linked to a solid phase support using a plurality of amino acids and at least one polyamino acid monomer, , where the linear peptide has a protecting group on one or more amino acids and/or at least one polyamino acid unit. In some embodiments, the synunit has at least one amine group that is acetylated and at least one carboxylic acid protecting group.

In some embodiments, the solid phase support is selected from the group consisting of Wang resin, Trityl resin, and Rink resin.

In some embodiments, the solid phase support has a weight of about 0.10 mmol/g, about 0.20 mmol/g, about 0.30 mmol/g, about 0.40 mmol/g, about 0.50 mmol/g, about 0.60 mmol/g, about 0.70 mmol/g, and has a loading of about 0.80 mmol/g, about 0.90 mmol/g, or about 1.00 mmol/g. In some embodiments, the solid phase has a loading of about 0.70 mmol/g. In some embodiments, the solid phase has a loading of about 0.90 mmol/g.

In some embodiments, the polyamino acid monomer is a compound represented by the formula:

where P ² is an amine protecting group; P ³ is a carboxylic acid protecting group; P ⁴ is a thiol protecting group.

In some embodiments, the protecting group is fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), trityl, methyl, ethyl, tert-butyl, allyl, 2,4 -Dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), benzyl (Bn), tert-butyldimethylsilyl, allyloxycarbonyl (alloc), tert-butyloxycarbonyl, acetamidomethyl (Acm) ), 3-nitro-2-pyridine sulfenyl (NPYS) and 2-pyridine-sulfenyl (Pyr).

In some embodiments, the amine protecting group P ² is selected from the group consisting of fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), and carboxybenzyl (Cbz). In some embodiments, P ² is a 9-fluorenylmethoxycarbonyl (Fmoc) protecting group.

In some embodiments, the carboxylic acid protecting group P ³ is the group consisting of methyl, ethyl, tert-butyl, allyl, 2,4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), benzyl (Bn). is selected from In some embodiments, P ³ is an allyl protecting group.

In some embodiments, P ⁴ is a trityl protecting group.

In some embodiments, the subunits of the polyamino acid monomer have a D-configuration, e.g., the monomer is a D-enantiomer. In some embodiments, polyamino acid monomers with subunits in the D-configuration can be represented by the formula:

.

.

In some embodiments, the subunits of the polyamino acid monomer have the L-configuration, for example, the monomer is an L-enantiomer. In some embodiments, polyamino acid monomers with subunits in the L-configuration can be represented by the formula:

.

.

In some embodiments, the subunits of the polyamino acid monomer have both the D-configuration and the L-configuration.

In some embodiments, the amino acid side chains of linear peptides have protecting groups. In some embodiments, the amino acid side chain protecting group is tert-butyl (tBu), trityl (Trt), allyl, cyclohexyl, 2-phenylisopropyl, acetamidomethyl (Acm), benzyl (Bzl), 4-methylbenzyl. (4-MeBzl), 4-methoxybenzyl (4-MeOBzl), 9-fluorenylmethyl (Fm), tert-butylthio (t-Buthio), 4-methoxytrityl (Mmt), xanthyl ( Xan), 2,6-dichlorobenzyl (2,6-Cl ₂ Bzl) and 2-bromobenzylcarbonate (2-BrZ). In some embodiments, the amino acid side chain protecting group is tert-butyl (tBu) or trityl (Trt).

In some embodiments, the amino acid side chains of the linear peptide with protecting groups on the side chains are Cys ₁ , Glu ₃ , Cys ₅ , Cys ₆ , Asn ₇ , Tyr ₁₁ , and Cys ₁₃ of SEQ ID NO:1.

In some embodiments, the plurality of amino acids and monomers are coupled by a carbodiimide-mediated reaction or with a non-carbodiimide coupling agent: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazole. [4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate Lophosphate (HBTU), 1H-benzotriazolium 1-[bis(dimethyl-amino)methylene]-5-chloro-hexafluorophosphate (1-),3-oxide (HCTU), O-(benzotriazole -1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7 -azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), or propanephosphonic anhydride (T3P), coupled by a reaction mediated by the linear peptide of step (i) forms.

In some embodiments, at least one amino acid from a plurality of peptides and/or monomers is coupled by a carbodiimide-mediated reaction to form the linear peptide of step (i). In some embodiments, the carbodiimide consists of diisopropylcarboximide (DIC), dicyclohexylcarbodiimide (DCC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). selected from the group. In some embodiments, the carbodiimide is DIC.

In some embodiments, the carbodiimide-mediated reaction further comprises an antioxidant. In some embodiments, the antioxidant is a soluble thiourea or thiol compound. In some embodiments, the antioxidant is 1,3-diisopropyl-2 thiourea (DITU) or dithiothreitol.

In some embodiments, at least one amino acid is coupled by a non-carbodiimide coupling agent. In some embodiments, the cyclization coupling reaction is mediated by a non-carbodiimide coupling agent. In some embodiments, the non-carbodiimide coupling agent used in the cyclization coupling reaction is HATU.

The linear peptide of step (i) may be referred to in this application as a “linear 13-mer” and may be represented by the formula:

,

,

에 의해 나타내어진다. 일부 실시양태에서, 밑줄친 아미노산의 측쇄 중 하나 이상이 보호된다. 일부 실시양태에서, 모든 밑줄친 아미노산의 측쇄는 보호된다.(SEQ ID NO: 2), wherein the cystathionine thioether side chain bridge -CH ₂ -CH ₂ -S-CH ₂ - is

It is expressed by . In some embodiments, one or more of the side chains of the underlined amino acids are protected. In some embodiments, the side chains of all underlined amino acids are protected.

After the linear peptide is formed, (ii) the protecting groups from the carboxylic acid protecting group of the synthetic unit and the amine group of the N-terminus of the linear peptide are removed to form a partially unprotected solid phase with an unprotected amine group and an unprotected carboxylic acid group. Forms a support-bound peptide. In some embodiments, deprotection is achieved using Pd(PPh ₃ ) ₄ and 1,3-DMBA in DMF.

In some embodiments, the partially unprotected solid phase support-bound peptide can be represented by the formula:

.

.

(SEQ ID NO: 3) After partial deprotection, (iii) the unprotected amine group and the unprotected carboxylic acid group are coupled to form a cyclized solid phase support-linked peptide.

In some embodiments, the cyclized solid phase support-linked peptide can be represented by the formula:

.

.

(SEQ ID NO: 4) The cyclized solid phase support-bound peptide is (iv) cleaved from the solid phase support to produce a cyclization protected peptide.

In some embodiments, linear peptides are cleaved from the resin via mild acid treatment. In some embodiments, mild acid treatment preserves the side chain protecting groups and the protecting groups of the polyamino acid monomer. In some embodiments, the dilute acid treatment is a weak acid solution, such as trifluoroacetic acid (TFA). In some embodiments, the dilute acid solution is a trifluoroacetic acid (TFA) solution. In some embodiments, the dilute acid solution is 1% trifluoroacetic acid (TFA) in dichloromethane (DCM) solution.

Once the cyclization protected peptide is cleaved from the resin, the peptide is (v) globally deprotected to yield a globally deprotected peptide. In some embodiments, overall deprotection step (v) comprises the addition of a cocktail comprising at least ammonium iodide (NH ₄ I). In some embodiments, overall deprotection step (v) comprises the addition of a cocktail comprising at least ammonium iodide (NH ₄ I) and triisopropylsilane.

Once the cyclized peptide is globally deprotected, the peptide is (vi) folded to form one or more additional bridges to form Ac-Cys ₁ Cth ₂ Glu ₃ Leu ₄ Cys ₅ Cys ₆ Asn ₇ Val ₈ Ala ₉ Cys ₁₀ Tyr ₁₁ Gly A synthetic peptide of ₁₂ Cys ₁₃ (SEQ ID NO: 1) is obtained.

As used herein, “folding” and “oxidation” may refer to the same step in which folding is achieved through oxidation of the cysteine residue of SEQ ID NO:1. In some embodiments, folding step (vi) is achieved via iodine- or alkali-mediated oxidation. In some embodiments, the alkali-mediated oxidation is dimethylsulfoxide (DMSO)- or N-methyl-2-pyrrolidone (NMP)-mediated oxidation.

In some embodiments, the synthetic peptide contains covalent bonds between the following amino acid residues of the synthetic peptide: Cys ₁ and Cys ₆ , Cth ₂ and Cys ₁₀ , and Cys ₅ and Cys ₁₃ . In some embodiments, the covalent bonds between Cys ₁ and Cys ₆ , and Cys ₅ and Cys ₁₃ are disulfide bonds. In some embodiments, the covalent bond between Cth ₂ and Cys ₁₀ is a thioether bond.

After folding of the overall deprotected peptide, the synthetic peptide of SEQ ID NO: 1 is purified.

In some embodiments, the synthetic peptide of SEQ ID NO: 1 can be represented by the formula:

.

.

(SEQ ID NO: 1)

Also described herein are methods for preparing synthetic peptides of Formula I:

The method includes the following steps:

(i) coupling the C-terminal resin-bound Tyr-Gly-Cys peptide with a protected amino acid side chain to a polyamino acid monomer of formula (II):

(here:

P ² is an amine protecting group;

P ³ is a carboxylic acid protecting group;

P ⁴ is a thiol protecting group)

Steps to form a resin bound peptide of formula III:

(ii) removing the P ² protecting group of Formula III to obtain a resin bound peptide of Formula IV with an unprotected amine group:

(iii) coupling P ² -alanine to the resin bound peptide of Formula IV via the free amine group of Formula IV to form the resin bound peptide of Formula V:

(iv) removing the P ² protecting group of formula V to obtain a free amine group, followed by coupling the free amine group to the P ² -amino acid,

wherein the side chain of the amino acid can be protected;

(v) repeating step (iv) five more times to form a resin bound peptide of formula (VI):

wherein at least one amino acid side chain is protected;

(vi) removing the P ² protecting group and the P ³ protecting group to obtain a free amine group and a free carboxylic acid group;

(vii) coupling the free amine group and the free carboxylic acid group to obtain a cyclized peptide of formula VII:

(viii) cleaving the peptide of formula VII from the resin to obtain a cyclized peptide;

(ix) globally deprotecting the cyclized peptide to obtain a globally deprotected peptide; and

(x) folding the globally deprotected peptide by forming two disulfide bonds to obtain the synthetic peptide of formula (I).

In some embodiments, the Glu, Cys, Cys, Asn, Gly, and Cys residues of Formula VI have side chain protecting groups. In some embodiments, the amino acid side chain protecting group is tert-butyl (tBu), trityl (Trt), allyl (All), cyclohexyl, 2-phenylisopropyl, acetamidomethyl (Acm), benzyl (Bzl), 4 -Methylbenzyl (4-MeBzl), 4-methoxybenzyl (4-MeOBzl), 9-fluorenylmethyl (Fm), tert-butylthio (t-Buthio), 4-methoxytrityl (Mmt), It is selected from the group consisting of xanthyl (Xan), 2,6-dichlorobenzyl (2,6-Cl ₂ Bzl) and 2-bromobenzylcarbonate (2-BrZ). In some embodiments, the amino acid side chain protecting group is tert-butyl (tBu) or trityl (Trt).

In some embodiments, P ² is a protecting group selected from the group consisting of fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), and allyloxycarbonyl (Alloc). In some embodiments, P ² is a fluorenylmethoxycarbonyl (Fmoc) protecting group.

In some embodiments, P ³ is methyl, ethyl, tert-butyl, allyl (All), trityl, 2,4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), and benzyl (Bn). It is a protecting group selected from the group consisting of: In some embodiments, P ³ is an Allyl (All) protecting group.

In some embodiments, P ⁴ is acetamidomethyl (Acm), tert-butyl (t-But), 3-nitro-2-pyridine sulfenyl (NPYS), 2-pyridine-sulfenyl (Pyr), and trityl. It is a protecting group selected from the group consisting of (Trt). In some embodiments, P ⁴ is a trityl protecting group.

Example

The following examples are merely illustrative of the invention and are in no way intended to limit the scope of the invention, since many modifications and equivalents encompassed by this disclosure will become apparent to those skilled in the art upon reading this disclosure. should not be construed as limiting.

abbreviation

AA: amino acid

AAA: amino acid analysis

Ac: Acetyl

AcOH: Acetic acid

All or Allyl: 2-propenyl

API: Active Pharmaceutical Ingredient

BB: (Fmoc-Cys ¹⁰ *[Ac-Cys(Trt)-Hcys(S*)-OAll]-OH) with the following structure:

.

.

C18: Silica gel C18

DBU: 1,8-diazabicyclo[5.4.0]undece-7-ene

DIC: diisopropylcarbodiimide

DIPEA: diisopropylethylamine

DITU: 1,3-diisopropyl-2-thiourea

1,3-DMBA: 1,3-dimethylbarbituric acid

DMF: N,N-dimethylformamide

DMSO: dimethyl sulfoxide

DTT: dithiothreitol

EtOH: Ethanol

eq: equivalent

Fmoc: 9-fluorenylmethoxycarbonyl

GC: gas chromatography

GSH/GSSG: glutathione (red./ox.)

HATU: 1H-1,2,3-triazolo[4,5-b]pyridinium, 1-[bis(dimethylamino)methylene]-, 3-oxide, hexafluorophosphate (1-) (1: One)

HDPE: High-density polyethylene

HPLC: High Performance Liquid Chromatography

IPA: isopropanol

LC: liquid chromatography

MeCN: Acetonitrile

MeOH: methanol

MTBE: Methyl tert-butyl ether

MS: mass spectrometry

Mw: molecular weight

NH ₄ OAc: Ammonium acetate

NMM: N-methylmorpholine

NMP: N-methylpyrrolidone

NMR: nuclear magnetic resonance

Oxyma: Ethyl (hydroxyimino) cyanoacetate

PPh ₃ : Triphenylphosphine

SEC: Size Exclusion Chromatography

SPPS: solid phase peptide synthesis

tBu: tert-butyl

TFA: trifluoroacetic acid

Thz: thiazolidine

Thz(Me) ₂ : 2,2-dimethylthiazolidine

TIS: Triisopropylsilane

Trt: Triphenylmethyl

Trt-H: Triphenylmethane

UV: ultraviolet rays

Example 1

Method for preparing synthetic peptide of SEQ ID NO: 1

The peptide of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof was prepared according to Good Manufacturing Practice (GMP) regulations.

matter

Fmoc-D-Cys(Trt)-OH (>97.0%, HPLC) and TFA (>99.0%, HPLC) for HPLC analysis were purchased from Sigma-Aldrich. Fmoc-Thz-OH (99.6%, HPLC) and Fmoc-Thz(Me) ₂ -OH (>99.3%, HPLC; 99.7% ee) were obtained from PepTech. BB (>98.5%, HPLC) was custom synthesized. The identity of the BB and L-enantiomers was confirmed by NMR and chiral analysis. DITU from Molekula, DMSO (pa) from Riedel-de Hahn, and O-methylhydroxylamine hydrochloride from Merck were used. GSH and GSSG were obtained from Sigma-Aldrich. All other reagents, amino acid derivatives, 2-CTC resin, and solvents were taken from the warehouse. All materials were used as received. Purified water was used.

equipment

synthesis equipment

For small-scale SPPS and cleavage/deprotection, special stopper syringes with filters were used. The syringe was automatically shaken with its contents at ambient temperature (20-25° C.).

Larger scale SPPS utilized a jacketed reactor with an overhead stirrer. The temperature was controlled in the jacketed reactor using a cryostat (Julabo).

Cleavage/deprotection starting from >5 g peptide resin was prepared in glassware or closed HDPE bottles.

Iodine-mediated oxidation (disulfide formation) was accomplished with the assistance of a syringe pump and suitable Teflon tubing.

Otherwise, standard laboratory equipment/tools were used for synthesis work.

Occasionally, a Beckman centrifuge (3750-4000 rpm, 10 minutes) was used for small-scale precipitation of cleaved/deprotected peptides.

analysis equipment

HPLC was performed using an Agilent system equipped with an XSelect Peptide 130 A 150 x 4.6 mm 2.5 μ column at 30°C. Mobile phase A: 0.1 % TFA(aq); Mobile phase B: 0.1% TFA (MeCN)

LC/MS used the same column on a Thermo Scientific Vanquish Horizon UHPLC coupled to an ESI Thermo Q-Exactive MS spectrometer. Chromeleon software was used for HPLC/MS evaluation.

Chiral AAA is C.A.T. GmbH & Co. Manufactured by C.A.T. GmbH & Co. Chromatographie and Analysentechnik KG (Tübingen, Germany).

NMR analysis was performed by RED GLEAD DISCOVERY AB, Medicon Village (Lund, Sweden 223 81).

peptide synthesis

SPPS

Figure 1 shows a flow diagram of the SPPS synthesis of the solid-bound 13-mer.

Standard protocols were used. All peptide resins were dried and stored in a freezer.

L- or D-H-Cys-2-CT resin

L- or D-Cys was attached to the 2-CTC resin for about 5 hours at 20-25°C in a SPPS reactor with a glass filter, and the remaining coupling sites on the resin were capped with methanol. After draining/washing, the Fmoc group was removed by treatment with 20% (v/v) piperidine in DMF (2 x 10 min). The resin was washed with DMF and then with isopropanol until a negative chloranyl test indicating removal of piperidine and finally dried under vacuum at 20-25°C for 1-2 days.

_L -H-Cys-2-CT resin; 102.0 g, 0.42 mmol/g (triphenylmethane)

_D -H-Cys-2-CT resin; 14.5 g, 0.40 mmol/g (triphenylmethane)

Fmoc(7-13)-2-CT Resin

For the L-Cys ₁₃ version, a jacketed reactor was used. The reaction was carried out at 20°C (T _jacket ), 20-25°C for _D -Cys ₁₃ .

Coupling was done in either DMF or NMP. For Cys ₁₃ loading, approximately 2.0 equivalents of AA derivatives were used, except for BB (1.5 equivalents). About 2.2 equivalents of Oxima/DIC, and about 0.2 equivalents of DITU were used. Glycine was preactivated for 1 hour. Additional DIC (about 2.2 equivalents) was generally added after some time. Total coupling time was typically 2-3 hours, and for BB was approximately 5 hours.

Glycine was preactivated to accelerate Gly coupling and thus avoid unintended dissociation of Cys from the acid-sensitive 2-CT resin by weakly acidic oximas or amino acids, potentially leading to endo- and des-Cys.

After each coupling, the reactor was drained and the resin was washed with DMF. When the synthesis was stopped overnight, the peptide resin was stored in the reactor (5-10°C). The operating temperature was increased before the synthesis continued.

The Fmoc group was removed by treatment with 20% (v/v) piperidine in DMF (2 x 10 min) and the resin was washed thoroughly until a negative chloranyl test, indicating complete removal of piperidine.

When the sequence was complete, it was washed with DMF and isopropanol. The resin was then dried under vacuum at 20-25°C for 1-2 days.

One of Cys _{(5 or 6)} was incorporated as a pseudoproline to facilitate cyclization by the cis-directed conformation. Suitable derivatives selected may be Fmoc-L-Thz-OH (CAS number [133054-21-4]) and Fmoc-L-Thz(Me ₂ )-OH (CAS number [873842-06-9]). Thz may replace Cys(Trt) at position 6 or 5, or both positions. Accordingly, four different Fmoc(3-13)-2-CT resin peptides were synthesized as shown below:

Yields are based on theoretical product weight.

Ring closure/lactamization

Fmoc(3-13)-2-CT resin was swollen with DMF in a syringe. After draining, Fmoc was removed with 20% piperidine (> 10 min + > 20 min). The resin was washed thoroughly until a negative chloranyl test was obtained. The allyl ester of Hcys was converted to the acid overnight using about 10 mol% Pd(PPh ₃ ) ₄ and about 10 equivalents of 1,3-DMBA in DMF. The resin was drained, washed with DMF, and cyclized using about 2 equivalents of HATU and 2.7 equivalents of NMM. The Kaiser test showed complete response within 4-5 hours. Final washing and drying were performed as described above.

Cleavage/deprotection

Assay scale cleavage/deprotection was performed in syringes as described below. The TFA solution was filtered into ice-cold (-18°C) diethyl ether. The precipitate was centrifuged, the supernatant was decanted, the residue was triturated with diethyl ether and the solid was centrifuged again.

Preparative scale (Ac-Cys(H)-Hcys(S1-Glu-Leu-Cys(H)-Cys(H)-Asn-Val-Ala-Cys*-Tyr-Gly-Cys(H)-OH):

Cyclic peptide resin Cys _{5 and 6} (Trt) (5.41 g) was mixed with 1.8 g DTT, 1.7 g NH ₄ I, 4.5 mL TIS, 1.8 mL water, and 50 mL TFA. The mixture was stirred for 3 hours. The resin was filtered and washed with 2 x 15 mL TFA. The combined filtrates were cooled (10°C), ice-cold (-18°C), and diethyl ether (360 mL) was added in portions over 10-15 minutes with stirring (T. 23°C). Stirring was continued at 0°C for approximately 5 minutes. The solid was filtered (16-40 μm) and washed with diethyl ether (2 x 100 mL). It was then dried at 20-25°C under vacuum for 2 days. This gave 1.80 g of crude cyclic peptide (free-SH).

Oxidation (formation of S-S bridges)

These experiments were obtained from Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Cys(Trt)-Asn(Trt)-Val-Ala-BB-Tyr(tBu)-Gly-Cys(Trt)-2CTC-resin. was performed with crude cyclic peptide (free-SH). All oxidation took place at 20-25°C.

Iodine oxidation

A solution of crude cyclized peptide in DMSO (10 g/L) was prepared. The solution was diluted 10-fold with 20% MeCN (aqueous) and became slightly cloudy (pH ca. 4). Iodine in MeCN (1% w/v) was added dropwise to this solution over approximately 1 hour until the yellow/brown color remained. Iodine was quenched with aqueous ascorbic acid (0.5 M). The pH was raised to 7-7.5 using 3.5% NH ₃ (aq). GSH/GSSG was added at different time points (1-3.5 mM).

DMSO oxidation

A clear solution was prepared with DMSO. These were diluted with 30% DMSO using water (pH approximately 4) to a concentration of 1 to 3.3 g/L. This slightly cloudy mixture was stirred at 60° C. or the pH was adjusted to 7.5-8 using 3.5% NH ₃ (aq.). At the latter pH, the solution became completely clear again. GSH/GSSG was added optionally.

NMP oxidation

A clear solution of 0.5 g crude material/mL NMP was prepared.

Results and Discussion

H-Cys(Trt)-2-CT and Fmoc(7-13)-2-CT resins

A Cys loading of 0.5 mmol/g resin was targeted. Consumption of Fmoc-Cys(Trt)-OH was followed by HPLC of the reaction solution. The reaction was stopped by washing at a stage (~5 hours) when 54 mmol, corresponding to approximately 0.6 mmol/g, had been incorporated. The loading was found to be 0.42 mmol/g.

From this Cys resin, we prepared the intermediate Fmoc(7-13) resin, which is common to all (3-13) candidates proposed for ring closure on the resin.

Based on Fmoc loading (0.199 mmol/g), the yield of Fmoc(7-13)-2-CT resin (61.9 g) was 81.6%. The purity of Fmoc(7-13)OH cleaved without side chain protection was approximately 85% (as determined by HPLC).

It may be possible for some peptides to be cleaved by the persistent action of the weakly acidic oxima because the ester linkage to 2-CT is acid sensitive. However, treatment of Fmoc(7-13)-2-CT resin with about 3 equivalents of Oxima in DMF for 21 hours (20-25°C) did not cause loss of peptides.

Although DMF was used throughout, NMP was also tested when performing the coupling. The latter solvent did not provide improvement.

DH-Cys(Trt)-2-CT (0.40 mmol/g) and [DH-Cys ₁₃ (Trt)]Fmoc(7-13)-2-CT (0.175 mmol/g) resins were mixed with the L-Cys ₁₃ version. It was prepared in the same way.

Fmoc(3-13)-2-CT resin

Syringe synthesis of Fmoc(3-13)-2-CT peptide was performed from 7.5 g Fmoc(7-13) resin each. Coupling was performed using Oxima/DIC for 3-5 hours, with additional DIC added after approximately 2 hours.

Yields are based on theoretical product weight.

After final washing/drying, a small sample was cleaved/deprotected and subjected to further analysis. Best results were observed using Cys(Trt) at both the 5 and 6 positions. Coupling to already incorporated Thz was slower (4-5 hours). The Thz(Me) ₂ variant lacks Fmoc-Gly-Leu.

Overcoming the resistance to continuous coupling of H-Thz(Me ₂ ) ₅ -Cys(Trt)-Asn(Trt)-Val-Ala-BB-Tyr(tBu)-Gly-Cys(Trt)-2-CT resin In an attempt to do this, it was again swollen in DMF and treated with HATU/NMM and Fmoc-Leu-OH overnight at 30°C. Activated Fmoc-Leu-OAt is formed, which is believed to be more reactive toward hindered amines. Unfortunately, this was not successful.

The estimated yield of the baseline Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Cys(Trt)-Asn(Trt)-Val-Ala-BB-Tyr(tBu)-Gly-Cys(Trt)-2CT resin is H -Cys(Trt)-2-CT resin was 48% (based on Fmoc loading and purity of (7-13) and (3-13) intermediates.

Ring closure (lactamization) on resin

Cyclization first removes Fmoc from the (3-13) resin, followed by Pd-catalyzed deallylation of Hcys for 4-5 h, and HATU assisted cyclization (coupling of -Hcy-OH to H-Glu-) It was done by. The reaction steps were carried out with the following resins:

Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Cys(Trt)-Asn(Trt)-Val-Ala-BB-Tyr(tBu)-Gly-Cys(Trt)-2CT Resin (Baseline Resin)

Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Cys(Trt)-Asn(Trt)-Val-Ala-BB-Tyr(tBu)-Gly-D-Cys(Trt)-2CT Resin

Fmoc-Glu(OtBu)-Leu-Thz ₅ -Cys(Trt)-Asn(Trt)-Val-Ala-BB-Tyr(tBu)-Gly-Cys(Trt)-2CT Resin

Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Thz ₆ -Asn(Trt)-Val-Ala-BB-Tyr(tBu)-Gly-Cys(Trt)-2CT Resin

Fmoc-Glu(OtBu)-Leu-Thz ₅ -Thz ₆ -Asn(Trt)-Val-Ala-BB-Tyr(tBu)-Gly-Cys(Trt)-2CT Resin

Among all-L analogues, the baseline resin produced the purest lactam, but the Kaiser test did not show any significant difference in degree of cyclization. Recall that the purity of the linear Thz resin was somewhat lower compared to the baseline resin due to incomplete coupling.

The estimated yield of the baseline resin was 32% (on H-Cys(Trt)-2-CT resin) based on the Fmoc/Trt loading and purity of the (7-13), (3-13), and (1-13) intermediates. It was about).

From the baseline resin (5.41 g), the lactams shown below were obtained in an amount of 1.80 g after cyclization and then cleavage/deprotection/precipitation.

(락탐).

(lactam).

The measured content of D-Cys in the baseline lactam indicates that epimerization of Cys ₁₃ is at least not very significant.

Oxidation (formation of S-S bridges)

Oxidation is the final step.

Although the baseline Ac(1-13)-OH cyclization product is sufficiently soluble in DMF for HPLC analysis, preparing a 10 g/L solution appears to be difficult. It appears to be very insoluble in MeCN, 20-80% MeCN (aqueous, with or without AcOH), pure AcOH, 25% AcOH (aqueous), water, EtOH and MeOH. It is highly soluble in DMSO, and the solution is stable (HPLC) at 5-10°C, and even when kept for several days at 25°C. A 10-fold dilution in 20% MeCN (aqueous) slightly clouded the resulting mixture, but there was no peptide loss upon filtration (HPLC). It is readily soluble in NMP (10 g/L), but reacts immediately.

Alkaline DMSO oxidation was the simplest method. Lowering the crude concentration from 3 to 1 g/L gave virtually identical results; No increase in purity was observed compared to the 3 g/L experiment. Regardless of the crude lactam concentration, there was no change in the HPLC profile when the folding time was extended from 1 to 2 days.

It was not clear whether GSH/GSSG actually had an effect on folding upon iodine oxidation, or whether it was just the increased pH that changed the HPLC profile; At least in the case of alkaline DMSO oxidation, GSH-GSSG played no discernible role.

NMP as a solvent was not useful because the peptide reacted very quickly into an undefined mixture. The high reactivity is most likely due to NMP(5-OOH) forming from NMP upon aging under oxidizing conditions (air).

conclusion

Synthesize synthetic peptides of SEQ ID NO:1 using the strategy presented in Figure 1, e.g. the standard SPPS protocol for assembling amino acids into the desired sequence, followed by formation of a HATU auxiliary amide bond between Hcys and Glu into the ring. This can be done to provide macrocyclic, protected peptides on the resins shown below:

The estimated yield of this cyclic peptide on a resin based on H-Cys(Trt)-2-CT resin was 32%. This corresponds to a cyclization yield of 67%.

Analogues with Thz at the 5 or 6 position, or both, did not provide benefit. Coupling to already incorporated Thz requires more time to complete. Substitution with these Cys analogs will also require additional processing steps prior to building the disulfide bridge to remove the methylene bridge with aqueous MeONH ₂ to scavenge formaldehyde. They were not deprotected by TFA cocktail.

Another Cys derivative, Fmoc-Thz(Me) ₂ -OH, was also studied. This is readily converted to Cys by TFA deprotection, but when constructed into a peptide sequence, further coupling was not possible.

Therefore, the cyclization baseline resin shown above is the best choice. Without Thz in the intermediate peptide sequence, analytical methods to detect possible residual formaldehyde would be unnecessary.

Upon ring closure, the lactams shown below were obtained by cleavage/deprotection/precipitation (1.80 g from 5.41 g of baseline resin):

,

,

This can also be represented by the formula:

.

.

Oxidation, eg formation of disulfides from the lactams, was accomplished with iodine or in solutions containing DMSO. Correctly folded products were formed (the best choice so far was alkaline DMSO oxidation (HPLC/SEC)).

Other Embodiments

The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Additionally, it should be understood that all values are approximate and are provided for illustrative purposes.

All patents, patent applications, publications, product descriptions, and protocols are incorporated by reference throughout this application, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

Sequence list electronic file attached

Claims (33)

A method of preparing a synthetic peptide or a pharmaceutically acceptable salt thereof comprising the following steps:
(i) chemically synthesizing a linear peptide having a protected amine group at the N-terminus and a C-terminus bound to a solid phase support using a plurality of amino acids and at least one polyamino acid synthesis unit, wherein the linear peptide is Having a protecting group on one or more amino acids and/or at least one polyamino acid synthesis unit;
wherein the synthetic unit has at least one acetylated amine group and at least one carboxylic acid protecting group;
(ii) removing the protecting group from the carboxylic acid protecting group of the synthetic unit and the amine group of the N-terminus of the linear peptide, thereby producing a partially unprotected solid phase support-bound peptide having an unprotected amine group and an unprotected carboxylic acid group; forming step;
(iii) coupling the unprotected amine group and the unprotected carboxylic acid group to form a cyclized solid phase support-linked peptide;
(iv) cleaving the cyclized solid-phase support-bound peptide from the solid-phase support to produce a cyclization-protected peptide;
(v) globally deprotecting the cyclization protected peptide to obtain a globally deprotected peptide;
(vi) folding the generally deprotected peptide to form one or more additional cross-links to obtain a synthetic peptide; and
(vii) purifying the synthetic peptide;
wherein the synthetic peptide comprises the following amino acid sequence:
Ac-Cys ₁ Cth ₂ Glu ₃ Leu ₄ Cys ₅ Cys ₆ Asn ₇ Val ₈ Ala ₉ Cys ₁₀ Tyr ₁₁ Gly ₁₂ Cys ₁₃ (SEQ ID NO: 1);
wherein the synthetic peptide contains covalent bonds between the following amino acid residues:
a) Cys ₁ and Cys ₆ ,
b) Cth ₂ and Cys ₁₀ , and
c) Cys ₅ and Cys ₁₃ . The method of claim 1 further comprising the following steps:
Freezing the synthetic peptide from solution. 2. The method of claim 1, wherein the solid support is selected from the group consisting of Wang resin, Trityl resin and Rink resin. 2. The method of claim 1, wherein the solid support has a weight of about 0.10 mmol/g, about 0.20 mmol/g, about 0.30 mmol/g, about 0.40 mmol/g, about 0.50 mmol/g, about 0.60 mmol/g, about 0.70 mmol/g. , having a loading of about 0.80 mmol/g, about 0.90 mmol/g, or about 1.00 mmol/g. The method of claim 5, wherein the polyamino acid monomer is a compound of the formula:

here:
P ² is an amine protecting group;
P ³ is a carboxylic acid protecting group;
P ⁴ is a thiol protecting group. The method according to any one of claims 1 to 5, wherein the protecting group is fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), trityl, methyl, ethyl, tert-butyl, allyl, 2,4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm), benzyl (Bn), tert-butyldimethylsilyl, allyloxycarbonyl (alloc), tert-butyloxy Carbonyl, acetamidomethyl (Acm), 3-nitro-2-pyridine sulfenyl (NPYS) and 2-pyridine-sulfenyl (Pyr). The method according to claim 5 or 6, wherein P ² is a 9-fluorenylmethoxycarbonyl (Fmoc) protecting group. The method according to any one of claims 5 to 7, wherein P ³ is an allyl protecting group. The method according to any one of claims 5 to 8, wherein P ⁴ is a trityl protecting group. The method according to any one of claims 1 to 9, wherein the subunit of the polyamino acid synthesis unit has a D-configuration. The method according to any one of claims 1 to 9, wherein the subunit of the polyamino acid synthesis unit has an L-configuration. 12. The method according to any one of claims 1 to 11, wherein the subunits of the polyamino acid synthesis unit have both D-configuration and L-configuration. 13. The method according to any one of claims 1 to 12, wherein the plurality of amino acids and synthetic units are formed by a carbodiimide-mediated reaction or with a non-carbodiimide coupling agent: 1-[bis(dimethylamino)methylene]-1H -1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), (2-(1H-benzotriazol-1-yl)-1,1,3 ,3-Tetramethyluronium hexafluorophosphate (HBTU), 1H-benzotriazolium 1-[bis(dimethyl-amino)methylene]-5-chloro-hexafluorophosphate (1-),3-oxide ( HCTU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), benzotriazol-1-yloxytripyrrolidinophosphonium Coupled by a reaction mediated by hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), or propanephosphonic anhydride (T3P) ring to form the linear peptide of step (i). 14. The method according to any one of claims 1 to 13, wherein at least one amino acid from a plurality of peptides and/or synunits is coupled by a carbodiimide-mediated reaction to form the linear peptide of step (i). How to do it. 16. The method of claim 14 or 15, wherein the carbodiimide is diisopropylcarboximide (DIC), dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. (EDC). A method selected from the group consisting of (EDC). 16. The method of claim 15, wherein the carbodiimide is DIC. 19. The method according to any one of claims 15 to 18, wherein the carbodiimide-mediated reaction further comprises an antioxidant. 18. The method of claim 17, wherein the antioxidant is 1,3-diisopropyl-2 thiourea (DITU) or dithiothreitol. 19. The method according to any one of claims 1 to 18, wherein the cyclization coupling reaction is mediated by a non-carbodiimide coupling agent. 20. The method of claim 19, wherein the non-carbodiimide coupling agent is HATU. 21. The method according to any one of claims 1 to 20, wherein the cyclized peptide of step (iii) contains a thioether linkage. 22. The process according to any one of claims 1 to 21, wherein the overall deprotection step (v) comprises the addition of a cocktail solution with ammonium iodide (NH ₄ I). 23. The method according to any one of claims 1 to 22, wherein the folding step (vi) is achieved via iodine- or alkali-mediated oxidation. 24. The method of claim 23, wherein the alkali-mediated oxidation is dimethylsulfoxide (DMSO)- or N-methyl-2-pyrrolidone (NMP)-mediated oxidation. 25. The method according to any one of claims 1 to 24, wherein the covalent bond between Cys ₁ and Cys ₆ and Cys ₅ and Cys ₁₃ is a disulfide bond. A method for preparing a synthetic peptide of formula (I):

Method comprising the following steps:
(i) coupling the C-terminal resin-bound Tyr-Gly-Cys peptide with a protected amino acid side chain to a polyamino acid monomer of formula (II):

(here:
P ² is an amine protecting group;
P ³ is a carboxylic acid protecting group;
P ⁴ is a thiol protecting group)
Steps to form a resin bound peptide of formula III:

(ii) removing the P ² protecting group of Formula III to obtain a resin bound peptide of Formula IV with an unprotected amine group:

(iii) coupling P ² -alanine to the resin bound peptide of Formula IV via the free amine group of Formula IV to form the resin bound peptide of Formula V:

(iv) removing the P ² protecting group of formula V to obtain a free amine group, followed by coupling the free amine group to the P ² -amino acid,
wherein the side chain of the P ² amino acid can be protected;
(v) repeating step (iv) five more times to form a resin bound peptide of formula (VI):

wherein at least one amino acid side chain is protected;
(vi) removing the P ² protecting group and the P ³ protecting group to obtain a free amine group and a free carboxylic acid group;
(vii) coupling the free amine group and the free carboxylic acid group to obtain a cyclized peptide of formula VII:

(viii) cleaving the peptide of formula VII from the resin to obtain a cyclized peptide;
(ix) globally deprotecting the cyclized peptide to obtain a globally deprotected peptide; and
(x) folding the globally deprotected peptide by forming two disulfide bonds to obtain the synthetic peptide of formula (I). 27. The method of claim 26, wherein the Glu, Cys, Cys, Asn, Gly and Cys residues of Formula VI have side chain protecting groups. The method of claim 26 or 27, wherein P ² is selected from the group consisting of fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz) and allyloxycarbonyl (Alloc). Method of being a selected protecting group. 29. The method of claim 28, wherein P ² is a fluorenylmethoxycarbonyl (Fmoc) protecting group. 29. The method according to any one of claims 26 to 29, wherein P ³ is methyl, ethyl, tert-butyl, allyl, trityl, 2,4-dimethoxybenzyl (Dmb), 9-fluorenylmethyl (Fm) and benzyl (Bn). 31. The method of claim 30, wherein P ³ is an allyl protecting group. 32. The method according to any one of claims 26 to 31, wherein P ⁴ is acetamidomethyl (Acm), tert-butyl (t-But), 3-nitro-2-pyridine sulfenyl (NPYS), 2-pyridine. -a protecting group selected from the group consisting of sulphenyl (Pyr) and trityl (Trt). 33. The method of claim 32, wherein P ⁴ is a trityl protecting group.